| In our previous study, MALDI-TOF MS were used to detect two small peptide:e RF3 b and DRC3 f, which expressed differently between the liver cirrhosis,hepatic cell carcinoma, chronic hepatitis B. It was proposed that the two peptides may be involved in mechanism of the progression of chronic hepatitis and disease. Later it had been found that e RF3 b and DRC3 f may inhibit hepatic cell apoptosis and alleviate hepatic injury induced by concanavalin A.In consideration of a primary study results, in order to clarify role of e RF3 b and DRC3 f in chronic liver disease occurrence and development, our survey selects their effects on liver fibrosis as the breakthrough point, and research them on the influence of the hepatic stellate cell proliferation, apoptosis, and collagen expression, and explore the related mechanism of signal transduction further by cell vitro culture genetic and RNA interference technology at the cellular and molecular level. This topic includes the following three parts: PartⅠ: Inhibition of e RF3 b and DRC3 f to the Fibrosis-Related Factors Expression of HSC Induced by TGF-β1Objective: To investigate the influence and related factors expression induced by the e RF3 b and DRC3 f TGF- β 1 in hepatic stellate cells.Method: ①Cultured HSC LX-2 cells in vitro were selected as the experimental object. Different concentrations of TGF-β1,e RF3 b and DRC3 f stimulated HSC screening suitable dose of TGF-β1 by MTT respectively. ② Exogenous e RF3 b and DRC3 f amino acid polypeptide, plasmid- expression-vector p EGFP-C2-GSPT2/ p EGFP-C2-GSPT-111,which raised the gene level of p EGFP-C2-GSPT2/ p EGFP-C2- GSPT-111, and plasmid gene silencing expression vector p GPU6-GSPT2 sh RNA intervene HSC stimulated by TGF- β 1, testing the related genes m RNA expression level of fibrosis-ralated factors by RT- real time PCR, and the related protein expression by Western blot.Results:1 The proliferating vitality of HSC at every concentration in the TGF-β1 groups was higher than that in the controls(P<0.01). During promoting the proliferation the platform period appeared at 10ng/ml.2 Effects of exogenous e RF3 b amino-acid peptide on the fibrosis ralated factors expression of HSC induced by TGF-β1.(1) The concentration of e RF3 b amino-acid peptide had a positive correlation with the inhibitory rate of HSC, and their Pearson correlation coefficients were 0.926, 0.992, 0.946, 0.968(P=0.024, 0.001, 0.015, 0.007) at 4 time point respectively when its concentration distribute 0.1-100ng/ml the inhibitory platform period appeared at 100ng/ml concentration.(2) For GSPT2, the quality of the m RNA and protein expression of TGF group is less than that in the control group(numerically P=0.364, or P<0.001); and is more in TGF+ e RF3 b than that in TGF group(P=0.002). For TGF-β1, the expression of the m RNA and protein in TGF group is more than that in the control group(P<0.001), and is less in TGF + e RF3 b than that in TGF group(P<0.001). All these show that GSPT2 and TGF-β 1 have mutual antagonism effect in HSC.(3) For Col Ⅰ, Col Ⅲ, CTGF andα-SMA, the m RNA and protein expression of the four factors in TGF group is more than that in the control group(P<0.01), and is less in TGF+e RF3 b group than that in TGF group(P<0.01).3 Effects of p EGFP-C2-GSPT2 and p EGFP-C2-GSPT2-111 on the fibrosis-ralated factors expression of HSC induced by TGF- β1.(1) For GSPT2, the quality of the m RNA and protein expression in TGF group is less than that in the control group(P<0.05); and is more in TGF+ p EGFP-GSPT2/ p EGFP-GSPT2-111 than that in TGF group(P<0.01). For TGF-β1, the expression of the m RNA and protein in TGF group is more than that in the control group(P<0.001), and is less in TGF+ p EGFP-GSPT2 / p EGFP-GSPT2-111 than that in TGF group(P<0.05 or P=0.058 for protein expression of TGF+p EGFP-GSPT2).(2) For ColⅠ, ColⅢ, CTGF andα-SMA, the m RNA and protein expression of the four factors in TGF group is more than that in the control group(P<0.01), and is less in TGF+p EGFP-GSPT2/ p EGFP-GSPT2-111 group than that in TGF group(P<0.05).4 Effects of p GPU6- GSPT2 sh RNA on the fibrosis-ralated factors expression of HSC induced by TGF- β1.(1) For GSPT2, the quality of the m RNA and protein expression in TGF group is less than that in the control group(P<0.001); and is more less in TGF+p GPU6-GSPT2 sh RNA than that in TGF group(P<0.001). For TGF-β1, the expression of the m RNA and protein in TGF group is more than that in the control group(P<0.001), and is further more in TGF+p GPU6- GSPT2 sh RNA than that in TGF group(P<0.05, =0.006).(2) For Col Ⅰ, Col Ⅲ, CTGF and α-SMA, the m RNA and protein expression of the four factors in TGF group is more than that in the control group(P<0.01), and is less in TGF+p GPU6-GSPT2 sh RNA group much more than that in TGF group(P<0.05).5 Effects of exogenous DRC3 f amino-acid-peptide on the fibrosis-ralated factors expression of HSC induced by TGF- β1.(1) DRC3 f amino-acid peptide had a positive correlation with the inhibition rate of HSC(P<0.05)when its concentration among 0.1-100ng/ml, and their pearson correlation coefficients were 0.981, 0.975, 0.978, 0.978 at 4 time points respectively. Inhibitory platform period appeared at 100ng/ml concentration.(2) For TGF-β1, the expression of the m RNA and protein in TGF group is more than that in the control group(P<0.001), and is less in TGF + DRC3 f than that in TGF group(P=0.136, 0.021). All these show that DRC3 f and TGF- β 1 have slightly mutual antagonism effect in HSC.(3) For Col Ⅰ, Col Ⅲ, CTGF andα-SMA, the m RNA and protein expression of the four factors in TGF group is more than that in the control group(P<0.01), and is less in TGF+ DRC3 f group than that in TGF group(P<0.01). DRC3 f influenced the four fibrosis-ralated factors more slightly than e RF3 b amino-acid-peptide.Conclusion:1 The most suitable dose of TGF-β1 concentrations in stimulating HSC was 10ng/ml.2 e RF3 b and DRC3 f amino-acid-peptide were used to work on HSC at 100ng/ml using dose appropriately.3 TGF-β1 can activated HSCs,which were induced to pruduce Col I, Col III, CTGF, a-SMA. HSC fibrosis-related model activated by TGF-β1 was constructed successfully.4 GSPT2 and TGF-β1 have mutual antagonism effects, and e RF3b/ DRC3 f played a role in HSC by inhibiting TGF-β1 m RNA and protein expression.5 Exogenous e RF3 b amino-acid peptide, e RF3 b gene up- regulated and e RF3 b gene silenced proved from positive and negative that e RF3 b can inhibit the expression of Col Ⅰ, Col Ⅲ, CTGF and α-SMA through TGF-β1 pathway in HSC.6 Exogenous DRC3 f amino acid peptide has inhibiting effects the expression of ColⅠ, Col Ⅲ, CTGF and α-SMA through TGF-β1 pathway in HSC. Part Ⅱ: Effects of e RF3 b and DRC3 f on the Proliferation and Cell Cycle of HSC Induced by TGF-β1Objective: To investigate the mechanism and effects of e RF3 b and DRC3 f on the proliferation and cell cycle of HSC induced by TGF-β1Methods: e RF3 b amino-acid peptide and DRC3 f amino-acid peptide, gene-up-regulated plasmid expression vector p EGFP-C2-GSPT2 and p EGFP-C2-GSPT2-111, gene-silencing plasmid expression vector p GPU6- GSPT2 sh RNA intervene HSC stimulated by TGF-β1. The cell proliferation vitality was examined by MTT. PI marker flow cytometry was used to detect the cell cycle, RT-real time PCR examined the m RNA expression of the related genes.Results:1 Effects of e RF3 b on the proliferation and cell cycle of HSC induced by TGF-β1.(1) The proliferation vitality of HSC in the TGF-β1 groups was higher than that in the control(P<0.001), and was lower in the e RF3 b groups than that in the TGF-β1 group(P<0.01). The inhibition rate of 100ng/ml at two time points were 34.80% and 19.90% respectively.(2) Compared with the control group, the cells in G0/G1 phase decreased significantly(P<0.001) TGF group, and S and G2/M cells were significantly increased(P ≤ 0.001). Compared with TGF group, G0/G1 phase cells increased(P<0.001), S phase cells decreased(P≤0.001). G2/M did not change significantly in TGF+e RF3 b group(P=0.178, 0.323) at 24 and 48 h.(3) The cell proliferation index of 3 experimental group were 45.74%, 74.00%, 56.97% at 24h(F=100.712, P<0.001)and 29.00%, 55.30%, 34.88% at 48h( F=46.296, P<0.001)respectively,which in TGFgroup is bigger than that in control(P<0.001),and smaller in TGF+e RF3 b group than that in TGF group(P<0.001).(4)The m RNA expression levels of Cyclin D1 and CDK4 in TGF group was higher than that in control(P<0.001, =0.018),and lower in TGF+e RF3 b group than that in TGF group(P<0.001). P21 m RNA in TGF group was lower than in control group(P=0.001),and was higher in TGF+e RF3 b group than that in TGF group(P=0.004).2 Effects of p EGFP-C2-GSPT2 and p EGFP-C2-GSPT2-111 on the proliferation and cell cycle of HSC induced by TGF-β1.(1)The proliferation vitality of HSC in the TGF-β1 groups was higher than that in the controls(P<0.01), and lower in the TGF+p EGFP-GSPT2 / p EGFP-GSPT2-111 ompared with the TGF-β1 group(P<0.05). The inhibitory rates were: 24 h: 20.68% and 22.56%; 48 h: 11.66% and 16.87%.(2)Compared with the control group, the cells in G0/G1 phase was decreased(P<0.001), S phase cells increased significantly(P<0.001) in the TGF group. In TGF+p EGFP-GSPT2/ p EGFP-GSPT2-111 group,G0/G1 phase cells increased significantly(P<0.001), S phase cells decreased significantly(P<0.001) compared with TGF group.(3)The cell proliferation index of G0/G1, S and G2/M phases were 35.71%, 51.49%,34.84% and 35.53% at 48 h respectively(F=18.746, P=0.01):That in TGFgroup is bigger than in control(P<0.001),and is smaller in TGF+p EGFP-C2-GSPT2/p EGFP-C2-GSPT2-111 group than that in TGF group(P<0.001).(4)The m RNA expression of Cycin D and CDK in TGF were higher than that in control group(P<0.001), and were lower in TGF+p EGFP- GSPT2 / p EGFP-GSPT2-111 than that in TGF group(P<0.05). P21 m RNA in TGF were lower than that in control(P=0.002)and were higher in TGF+p EGFP-GSPT2/ p EGFP-GSPT2-111 than in TGF(P=0.048, 0.007).3 Effects of GSPT2 gene silencing on the proliferation and cell cycle of HSC induced by TGF-β1.(1) The cell proliferation vitality in TGF was higher than that in Control(P<0.001), and further higer in TGF+p GPU6-GSPT2 group than that in TGF group respectively at 24 and 48 hours(P=0.003, 0.001)(2) Compared with the control group, the cells in G0/G1 phase in TGF group was more decreased(P=0.001), S phase cells more increased(P<0.001). G2/M did not change significantly(P=0.319). Compared with TGF group, the cells in G0/G1 phase in TGF+p GPU6-GSPT2 sh RNA group decreased further(P=0.029), S phase cells increased(P=0.001), G2/M decreased(P<0.001), but the total number of cells in S+G2/M phase was increased.(3) The cell proliferation index of G0/G1, S and G2/M phases were 35.71%,51.49%,58.20% at 48 h respectively(F=47.928, P<0.01):That in TGFgroup was bigger than in control(P<0.01),and was bigger in TGF+ p GPU6-GSPT2 sh RNA group than in TGFgroup(P<0.05) further.(4)The m RNA expression in Cyclin D and CDK4 in TGF were higher than that in control(P<0.001),and had a further improvement in TGF+ p GPU6-GSPT2 sh RNA group than that in TGF group(P≤0.001). P21 m RNA in TGF was lower than that in the control group(P=0.001), and had a further decrease in TGF+p GPU6-GSPT2 sh RNA group than that in TGF group(P<0.001).4 Effects of DRC3 f on the proliferation and cell cycle of HSC induced by TGF-β1.(1) The proliferation vitality of HSC at 24 and 48 h increased(P<0.01)compared with the control, the DRC3 f groups decreased compared with the TGF group(P<0.01). The inhibition rate at 100ng/ml concentration were 34.13% and 21.12% at 24,4 8h respectively.(2) Compared with the control group, the cells in G0/G1 phase decreased(P<0.001) in TGF group, S and G2/M cells were increased significantly(P<0.001). Compared with TGF+DRC3f group and TGF group, G0/G1 phase cells increased(P<0.001), S phase cells decreased(<0.001),and G2/M phase cells remain original level(P=0.178).(3) The cell proliferation index of 3 experimental group were 45.74%, 74.00%, 52.23% at 24h(F=91.597, P<0.001)and 29.00%, 55.30%, 31.40% at 48h(F=47.816, P<0.001)respectively,and that in TGFgroup is bigger than in control(P<0.001),and smaller in TGF+ DRC3 f group than that in TGF group(P<0.001).(4) The m RNA expression of Cyclin D1 and CDK4 in TGF was higher than in the control(P<0.001), and was lower in TGF+DRC3f than that in TGF group(P=0.034, <0.001). P21 m RNA in TGF was lower than in the control(P=0.001), and was higher in TGF+DRC3f than that in the TGF group(P=0.034).Conclusion:1 e RF3 b amino-acid peptide, e RF3 b gene up-regulated and e RF3 b gene silenced aspects proved that e RF3 b has inhibitory effects on the proliferation of HSC induced by TGF-β1. At the same time e RF3 b reduced S cell cycle at which DNA synthesized, and arrest cell growth at period of G0/G1 phase. The mechanism is associated with the e RF3 b adjusting expression of Cyclin D1,CDK4 and p21 gene.2 DRC3 f amino-acid-peptide has inhibitory effects on the proliferation of HSC induced by TGF-β1. At the same time DRC3 f reduced S cell cycle at which DNA synthesized, and arrest cell growth at period of G0/G1 phase. The mechanism is associated by regulating the expression of Cyclin D1,CDK4 and p21 gene.3 Inhibition of proliferation, S cell cycle and DNA synthesis to HSC may be one of machaisms e RF3 b and DRC3 f resisted fibrotic factor. Part Ⅲ: Effects of e RF3 b and DRC3 f on Apoptosis and Migrating Vitality of HSC Induced by TGF-β1Objective: To study the effects and mechanism of e RF3 b and DRC3 f on apoptosis and migrating vitality of hepatic stellate cell induced by TGF-β1.Methods: e RF3 b and DRC3 f amino acid peptide, upregulate gene expression plasmid vector p EGFP-C2-GSPT2, p EGFP-C2-GSPT-111, expression of gene silencing vector p GPU6- GSPT2 sh RNA intervention HSC stimulated by TGF-β1 at 24 h and 48 h. PI marker flow cytometry was used to detect the apoptosis vitality, The migration ability of the cell was examined by scratch test. RT-real time PCR was used to detect related gene m RNA expression.Results:1 Effect of exogenous e RF3 b amino-acid peptide on apoptosis and migrating vitality of HSC induced by TGF-β1.(1) In 24, 48 h, the apoptosis rate of HSC in 3 experimental groups were: 0.56%, 4.66%, 0.72%; 1.66%, 13.35%, 2.71% respectively, and increased in TGF group than in the control group(P<0.001), and was significantly reduced in e RF3 b group than in the TGF group(P<0.001).(2) The expression of Bcl-2 m RNA in TGF group decreased than that in the control group numerically, but the difference was not statistically significant(P=0.147); and that in TGF+e RF3 b group increased than in the TGF group(P=0.043). The expression of Bax and Fas in TGF group increased than Control(P=0.011, 0.004); and that in TGF+e RF3 b group decreased than in the TGF group( P=0.004) or decreased numerically(P=0.456).(3) The relative migration distance in TGF-β1 group was higher than that in the control at same time(P=0.003, 0.001), and was lower in TGF+ e RF3 b group than that in the TGF-β1 group(P=0.018, 0.005). In 3 experimental groups relative expression level of α-SMA m RNA was higher than that in control(P<0.001), and was lower in TGF+e RF3 b group than that in TGF group(P=0.001).2 Effect of p EGFP-C2-GSPT2 and p EGFP-C2-GSPT2-111 to the apoptosis and migrating vitality of hepatic stellate cells.(1) The apoptosis rate in these 3 experimental groups were: 1.66%, 13.35%, 1.93%, 1.46% respectively. The apoptosis rate in TGF group increased than that in the control group(P<0.001), and decreased in TGF+p EGFP-GSPT2 /p EGFP- GSPT2-111 group than that in the TGF group(P<0.001).(2) The expression of Bcl-2 m RNA in TGF group decreased than that in the control group(P=0.108), and was increased in TGF+p EGFP-GSPT2/ p EGFP- GSPT2-111 group than that in the TGF group numerically(P=0.519, 0.303). The expression of Bax and Fas m RNA increased than that in the control group(P=0.030, 0.007), and was increased in TGF+p EGFP-GSPT2/ p EGFP-GSPT2-111 group than that in the TGF group numerically(Bax: P=0.297, 0.137; Fas: P=0.593, 0.408).(3) The relative migration distance in TGF-β1 group was higher than that in the control at same time(P=0.003,<0.001), and was lower in TGF+p EGFP-GSPT2/p EGFP-GSPT2-111 group than that in the TGF-β1 group(24h:P=0.021,0.009;48h:P<0.001). The relative expression level of α-SMA m RNA in TGF group was higher than that in the control(P<0.001),and was lower in TGF+e RF3 b group than that in TGF group(P<0.001).3 Effect of p GPU6 GSPT2 sh RNA on apoptosis and migrating vitality of hepatic stellate cells.(1) The apoptosis rate of 3 experimental groups were 1.66%, 13.35%, 5.53% respectively. The apoptosis rate increased significantly in TGF group than that in the control group(P<0.001), and was further increased in TGF+p GPU6-GSPT2 sh RNA group than that in the TGF group numerically(P=0.096).(2) The m RNA expression of Bcl-2 in TGF group decreased than in the control group(P=0.008), and further decreased in TGF+p GPU6-GSPT2 sh RNA group than in the TGF group(P=0.650). The expression of Bax and Fas in TGF group increased than in the control group(P=0.005, 0.001), and increased further in TGF+p GPU6-GSPT2 sh RNA group than that in the TGF group numerically( P=0.002, 0.766).(3) The relative migrating distance of TGF-β1 group was higher than that in the control at same time(P=0.001, P<0.001) and had a further increasement in TGF+p GPU6-GSPT2 sh RNA than TGF group(P=0.002,0.222). The α-SMA m RNA expression levels in TGF was higher than that in the control(P<0.001), and that in TGF+p GPU6- GSPT2 sh RNA had a further increasement than in TGF group numerically(P=0.009).4 Effects of exogenous DRC3 f amino acid peptide on the apoptosis and migrating vitality of HSC induced by TGF-β1?(1) At 24 and 48 h, the apoptosis rate of 3 experimental groups were: 0.56%, 4.66%, 0.91%; 1.66%, 13.35%, 3.08% respectively. The apoptosis rate in TGF group increased significantly than in the control group(P<0.001), and was significantly reduced in DRC3 f group than that in the TGF group(P<0.001).(2) The m RNA expression of Bcl-2 in TGF group has decreased than Control(P=0.208), meanwhile increased in TGF+DRC3f group than in TGF group numerically(P=0.204). The expression of Bax and Fas in TGF group increased than that in the control group(P=0.018, 0.004), and decreased in TGF+DRC3f group than that in the TGF group numerically(P=0.150,0.653).(3) The relative migration distance in TGF-β1 group was higher than that in the control at same time(P=0.002),and was decreased in TGF+DRC3f compared with TGF group(P=0.146, 0.023). The relative α-SMA m RNA expression in TGF was higher than in the control(P<0.001), and was lower in TGF+DRC3f than that in TGF group(P<0.001).Conclusion：1 Exogenous e RF3 b amino-acid peptide, e RF3 b gene up- regulated and e RF3 b gene silenced proved from positive and negative that e RF3 b has inhibitory effects on the apoptosis of HSC induced by TGF-β1. The mechanism may be that e RF3 b regulates the expression of Bcl-2, Bax and Fas m RNA level in HSC.2 e RF3 b has inhibitory effects on the migration vitality of HSC induced by TGF-β1 proved from positive and negative. The mechanism may be that e RF3 b inhibits the expression of α-SMA m RNA in HSC.3 Exogenous DRC3 f amino-acid peptide has an inhibitory effect on apoptosis induced by TGF-β1; Mechanism: DRC3 f adjusts the expression of Bcl-2,Bax and Fas m RNA level.4 DRC3 f amino acid peptide has inhibitory effect on migrating vitality induced by TGF-β1. DRC3 f inhibit the expression level of α-SMA in HSC.5 Inhibition of apoptosis and migrating vitality to HSC may be one of machaisms e RF3 b and DRC3 f resisted fibrotic factor. Conclusion:... |
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